Chlamydia species are obligate intracellular parasites that are important causes of a wide range of human diseases. Chlamydia species have a unique intracellular life cycle but understanding its details and the mechanisms of disease pathogenesis has been hampered by the difficulty in growing the organism and the lack of genetics. Our long term goal is to understand how Chlamydia causes disease in humans. Our short term goals are to identify host genes required for Chlamydial pathogenesis. In this new R01, we use the results of a novel forward genetic screen that employed genome-wide RNAi in Drosophila S2 cells to identify host factors required for successful infection. In previous work, we have established that C. trachomatis infection of Drosophila tissue culture cells mimics key aspects of initial Chlamydia-mammalian cell interactions. We have completed a primary and secondary screen which has identified ~125 host genes that affect binding, entry, and/or early vacuole formation. We propose comprehensive and complementary approaches to validate the role of these host genes during C. trachomatis infection of mammalian cells. In the first approach, we will validate and further study host genes whose function, inferred by homology, leads to obvious testable predictions based on pre-existing data ("harvesting the low lying fruit"). In the second approach, we will investigate host genes whose functions remain unknown but which have the potential to yield novel insights ("going for the unknown"). In each case, we will assess the role of the candidate gene in mammalian cell infections by performing RNAi-mediated gene inactivation in mammalian (HeLa) cells. We will confirm promising candidate genes using pharmacologic approaches, transfection of constitutively active or dominant negative alleles when available, or appropriate knock-out cells. We will determine at which, step each host molecule of interest is required, by quantifying how binding, entry, and intracellular development is affected upon depletion of the host genes. Finally, we will determine whether the host gene is required in an animal model of infection, using novel RNAi-based technologies. Together, these approaches will maximize the potential of this novel screen to systematically and comprehensively identify new host genes important in the pathogenesis of chlamydial infections. These findings will increase our basic knowledge of the pathogenesis of intracellular infections. In addition, they have the potential to identify new targets for the development of new therapeutic, diagnostic, and preventative therapies. Chlamydia species are an important cause of human diseases world-wide. This grant will discover what host genes are required for infection. This may allow the development of new drug and vaccine targets.